Project Summary Dysfunction of renal epithelial Cl- transport is associated with human diseases such as Bartter and Gitelman syndromes as well as in salt-sensitive hypertension. Mutations in WNK4 kinase have been linked to hypertension in pseudohypoaldosteronism type II (PHAII). PHAII-causing mutant WNK4 increases paracellular Cl- permeability and phosphorylates tight junction (TJ) protein claudins. Recently, we have found that claudin-7 plays a crucial role in regulating paracellular Cl- permeation and is a specific TJ target of WNK4 kinase. Claudin-7 knockout mice (Cln7-/-) display salt wasting and water loss phenotypes, suggesting the impairment of ion reabsorption in renal tubules. Our long- term goals are to understand TJ protein functions in kidneys and their contribution to ionic imbalance in human diseases such as hypertension. This project will test the hypothesis that claudin-7 is essential for TJ functions in renal epithelial cells and interacts with WNK4 in modulation of paracellular Cl- permeation. This application has three Specific Aims: (1) to characterize our recently generated Cln7-/- mice and determine whether claudin- 7 is essential in the formation of paracellular pores allowing Cl- permeation. We will use primary epithelial cells isolated from collecting duct (CD) of Cln7+/+ and Cln7-/- mice to determine their paracellular ion selectivity. We will transfect wild-type claudin-7 to determine if claudin-7 functions can be restored in Cln7-/- CD cells. We will also transfect the extracellular domain (ED) mutants of claudin-7 into Cln7-/- CD cells and LLC-PK1 cells with claudin-7 knockdown by RNAi to determine the role of claudin-7 ED in paracellular ion selectivity; (2) to investigate the regulation of claudin-7 mediated paracellular Cl- permeability by WNK4 kinase. We will determine if the expression and localization of WNK4 are altered in epithelia of distal nephron in Cln7-/- mice as well as in Cln7-/- CD cells. We will determine the changes in paracellular Cl- permeability by the expression of WNK4 and its PHAII-causing mutant in CD cells. Claudin-7 phosphorylation-null and -mimic mutants at WNK4 site will be transfected into Cln7-/- CD cells as well as LLC-PK1 cells to determine the role of WNK4 phosphorylation of claudin-7 on paracellular ion selectivity and (3) to investigate the roles of claudin-7 deletion in the development of salt-wasting and acute tubular necrosis (ATN) in Cln7-/- mouse kidney. TJ ultrastructure, barrier function, blood and urine ion concentration as well as plasma renin level will be compared at postnatal day 1, 4, and 7 Cln7-/- mice to determine how Cln7-/- phenotypes develop after birth. Newborn Cln7-/- mice will be subject to NaCl supplement to determine if it delays ATN phenotype and prolong the life of Cln7-/- mice. Project Narrative This project will test the hypothesis that claudin-7 is essential for tight junction functions in kidney epithelial cells and interacts with WNK4 in modulation of paracellular Cl- permeation. This project has three specific aims to (1) characterize our recently generated claudin-7 knockout (Cln7-/-) mouse line and determine whether claudin-7 is essential in the formation of paracellular pores allowing Cl- permeation; (2) investigate the regulation of claudin-7 mediated paracellular Cl- permeability by WNK4 based on our findings that claudin-7 interacts with and is phosphorylated by WNK4 and (3) investigate the roles of claudin-7 deletion in the development of salt-wasting and acute tubular necrosis in Cln7-/- mouse kidney.